Capture of target substances, such as biomolecules, on solid phases has been used for years in order to facilitate their subsequent identification and to study their manner of interacting with other substances. Typical interactions are those between proteins, such as between antigens and antibodies, hormones and receptors, biotinylated molecules and biotin binding proteins, and the like. One method of capture is generally designated as “blotting,” whereby the target substance is applied, directly or by transfer from another medium, to a membrane, such as nitrocellulose, polyvinyllidene difluoride (PVDF), or nylon.
Blotting is most frequently used in combination with known gel electrophoresis procedures, whereby the target substance, e.g., an antigen, is first separated on a gel from other substances, typically by the electrophoresis procedure commonly referred to as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The target substance is then transferred electrophoretically to a membrane. Subsequently, the target is associated, either directly or indirectly through an intermediate primary ligand, by an affinity interaction to a reporter probe, such as an antibody, having a label attached thereto which is capable of providing a detectable signal. Radioactive, calorimetric, fluorescent, or enzymatic labels are commonly employed to provide the detectable signals which, in turn, indicate the presence and/or location of the target molecule. Responses from radioactive and fluorescent labels are generally determined directly, while the response (luminescent, fluorescent, or colorimetric) elicited with enzymatic labels is indirect in that the detectable signal results from the action of the enzyme on an appropriate substrate.
Procedures for electrophoretic separation with subsequent blotting onto a membrane have historically been referred to in the literature as either Western blotting, Northern blotting or Southern blotting. Western blotting refers to the identification of antigens as target substances, while the latter two procedures refer to identification of target RNA and DNA sequences, respectively. More recently, a variation of Western blotting, referred to as Far Western blotting, has been used to characterize protein to protein interactions, other than antigen to antibody.
A drawback associated with Western type and other blotting techniques is that they require time consuming and cumbersome steps. These include transferring the target substance from the gel to the membrane on which the substance is immobilized and then blocking the membrane. Some of these steps, particularly the transfer and immobilization operations, may be detrimental to the protein being assayed. For example, a change in antigenic nature of a protein may prevent the corresponding antibody from binding and, therefore, detecting the target molecule. Also, the pattern obtained on a membrane when a crude lysate is transferred may not be a true representation, since smaller molecular weight proteins transfer more efficiently than larger molecular weight proteins. Additionally, some proteins simply do not transfer well and, therefore, are not represented on the membrane at all.
Thus, “in gel” procedures, in which detection is accomplished without removal of the target from the gel, have distinct advantages. And, while “in gel” techniques for detection of target molecules in polyacrylamide gel were reported even before the advent of the above described blotting techniques (Burridge, K. (1976) Proc. Natl. Acad. Sci., USA, 73, 4457–4461; Rosta, J. A.; Kelly, P. T.; and Cotman, C. W. (1977) Anal. Biochem., 80, 336–376; and Olden, K. and Yamada K. M. (1977) Anal. Biochem, 78, 483–490), the procedures were very time consuming, entailing lengthy fixing, incubation and wash steps, which generally took on the order of several days. Furthermore, “in gel” techniques have heretofore been considered not sufficiently sensitive for the detection of target molecules using large reporter probes, e.g., antigen-antibody interactions. The problem encountered is to obtain sufficient penetration of the probe or primary ligand into the three-dimensional gel to achieve the required association of the target and probe to permit detection at low concentrations of target.
Accordingly, a sought after objective is to provide an efficient “in gel” detection method which is sensitive and useful for the detection of biomolecules. And, it is with respect to this objective that the present invention is directed.